The inhibitors described below can be used for a number of aspartic proteases, such as HIV protease, renin, pepsin and cathepsin D. However, for ease of discussion, reference will be made to applying the described inhibitors to HIV protease. It will be well understood that optimization of inhibitors for renin, pepsin and cathepsin D may be required. The nature and scope of inhibitor modifications for such optimization will be readily apparent in view of the discussion below.
The human immunodeficiency virus (HIV) encodes an aspartic protease whose function is essential for proper virion assembly and maturation. Inactivation of HIV protease either by mutation or chemical inhibition leads to the production of immature, non-infectious viral particles. Accordingly, in attempts to find a drug for the treatment of AIDS, efforts have been directed to inhibitors of HIV protease.
HIV protease is unique in the family of aspartic proteases in that it is a homodimer which displays C.sub.2 symmetry about the active site. The dimer is made up of two identical sub-units each contributing an aspartate residue to form a single active site. Known inhibitors are designed to inactivate HIV protease by interaction with the carboxylic acid functional group of one or both of the aspartic acid residues in the active site.
In the absence of a substrate, a water molecule bridges the two carboxyl groups of the aspartic acid residues by hydrogen bonding to two oxygen atoms; one from each of the carboxyl groups. In the active form of the enzyme, the two carboxyl groups thus interact closely and share one proton between them. Because of this interaction, one of the carboxyl groups has a lower pK.sub.a value, typically about 1.5, while the other carboxyl group has a higher pK.sub.a, value, typically about 4.7.
Reduced amide inhibitors were among the earliest HIV protease inhibitors.
One of the more notable of these inhibitors is known as MVT-101. While the inhibitor does show activity against HIV protease, it is believed that the lack of functionality with the reduced amide (--CH.sub.2 --NH--) for hydrogen bonding to the aspartic acid residues is responsible for the moderate potency of the reduced amide inhibitors (Kempf, D. J. et al. Current Pharmaceutical Design 2:2:225-246; 1996).
Accordingly, most of the conventional inhibitors developed to date are the "so-called" transition-state analogs. Examples of transition-state analogs include statine-, hydroxyethylene- and hydroxyethylamine-containing inhibitors. These transition-state analog inhibitors share the common feature of a central hydroxyl group for hydrogen bonding to the carboxyl groups of the two aspartic acid residues in the active site.
The accepted mechanism of action for transition-state analog inhibitors is that the central hydroxyl group of the inhibitor replaces the water molecule that was bound in the active site of the enzyme in the absence of a substrate or inhibitor. The hydroxyl group acts as both a transition-state inhibitor and as a hydrogen bond donor-acceptor.
A common feature of hydroxyethylamine inhibitors is the presence of amine groups separated from the central hydroxyl group by 2 carbon atoms. No apparent interaction has been observed between the aspartic acid residues and the amine groups in the inhibitors (Huff, J. R. Medicinal Chemistry 34:8:2305-2314;1991).
In addition to the principal interaction between the central hydroxyl group and one or both of the carboxyl groups of the aspartic acid residues, there are a number of hydrogen bonds formed between the side chains of the inhibitor and sub-sites of the enzyme. The sub-sites of most interest in inhibitor design are termed the S.sub.1, S.sub.1 ', S.sub.2 and S.sub.2 ' sub-sites. Hydrogen bonding and other interactions between the atoms of the side chains and sub-sites helps stabilize the inhibitor in the active site of HIV protease (Wlodawer et al. Annu Rev Biochem 62:543-85; 1993).
Also, HIV protease has a pair of .beta.-hairpin flaps that cover the active site. These flaps interact with the substrate or inhibitor to tightly bind a water molecule. This interaction is illustrated in Huff, J. R. (ibid) as being hydrogen bonds formed by the flap IIe.sup.50 and IIe.sup.50 ' amide hydrogen atoms and the inhibitor carbonyl oxygen atoms on either side of a central hydroxyl group which hydrogen bonds to the aspartic acid residues. Thus, the water molecule bridges the inhibitor and the flaps. In the contracted conformation, the flaps form a pocketed hydrophobic tube shielding about 80% of the bound inhibitor from surrounding solvent (Huff, J. R., ibid).
One of the disadvantages of some of the known inhibitors is the poor pharmacokinetic properties and bioavailability of peptide analog inhibitors. High lipophilicity, high molecular weights, and the presence of numerous amide bonds contribute to less desirable pharmacokinetic properties and metabolic instability. Some researchers have therefore designed inhibitors in which an amide bond of a known tripeptide analog inhibitor has been replaced by an imidazole substituent, resulting in a substantial improvement of the pharmacokinetic properties and oral bioavailability of the inhibitor (Abdel-Meguid, S. S. et al. Biochemistry 33:39:11671-7; 1994).
Abdel-Meguid et al. disclose a hydroxyethylene tripeptide analog inhibitor in which the C-terminal carboxamide is replaced with imidazole to produce (2R,4S,5S,1'S)-2-phenylmethyl-4-hydroxyl-5-(tert-butoxycarbonyl)amino-6-ph enylhexanoyl-N-(1'-imidazo-2-yl)-2'-methylpropanamide. The central hydroxyl group interacts with the carboxyl groups of the active site aspartic acid residues. The imidazole replaced the carbonyl oxygen and nitrogen atoms of the C-terminal carboxamide that formed hydrogen bonds with the Asp.sup.29 .alpha.-amino group and the Gly.sup.48 carbonyl group of the HIV protease. The imidazole ring provides improved solubility, which may allow more efficient uptake of the inhibitor into the intestinal mucosa. It is also believed that the imidazole substitution favorably influences metabolic stability and clearance.
Disadvantages of inhibitors known to date include poor solubility, metabolic stability and bioavailability. Also, synthesis of these inhibitors is complicated due to the number of asymmetric centers in each compound.
Accordingly, there is a need for aspartic protease inhibitors that have the potential for improved in vivo performance, and which are comparatively more economical and easier to synthesize.